Human Natriuretic Peptide Research Articles

Allosteric activation of a spring-loaded natriuretic peptide receptor dimer by hormone.

Natriuretic peptides (NPs) are vasoactive cyclic-peptide hormones important in blood pressure regulation through interaction with natriuretic cell-surface receptors. We report the hormone-binding thermodynamics and crystal structures at 2.9 and 2.0 angstroms, respectively, of the extracellular domain of the unliganded human NP receptor (NPR-C) and its complex with CNP, a 22-amino acid NP. A single CNP molecule is bound in the interface of an NPR-C dimer, resulting in asymmetric interactions between the hormone and the symmetrically related receptors. Hormone binding induces a 20 angstrom closure between the membrane-proximal domains of the dimer. In each monomer, the opening of an interdomain cleft, which is tethered together by a linker peptide acting as a molecular spring, is likely a conserved allosteric trigger for intracellular signaling by the natriuretic receptor family.

Systematic screening of type B human natriuretic peptide receptor gene polymorphisms and association with essential hypertension.

C-type natriuretic peptide (CNP) dilates arteries, lowers blood pressure and inhibits proliferation of vascular smooth muscle cells via the type B natriuretic peptide receptor (NPRB). The CNP-NPRB system may play a crucial role in the development of cardiovascular disease. We recently determined the structure of the human NPRB gene. In the present study, our objectives are to identify the polymorphisms of the NPRB gene and investigate the association of this gene with essential hypertension (EH). We used the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique to study the NPRB gene polymorphism, and conducted an association study using a novel polymorphic marker. PCR-SSCP analysis of all 22 exons was done in 90 subjects, and abnormally-migrating bands were observed in the analyses of exon 11 and intron 18. Direct sequencing of these DNA fragments revealed the following sequence alterations: a C to T transition at nucleotide (nt) 2077 in exon 11 and a 9-bp insertion/deletion (I/D) in intron 18. PCR-restriction fragment length polymorphism analysis (PCR-RFLP) was developed to detect the C2077T transition. PCR-RFLP analyses of healthy subjects revealed that the C2077T polymorphism had complete linkage to GT repeats in intron 2 reported previously. The I/D polymorphism was identified by polyacrylamide gel electrophoresis, and it was not linked to any known polymorphic alleles of this gene. Therefore, the possible association between the I/D polymorphism and EH was investigated. A total of 123 individuals with EH and 123 age-matched normotensive control subjects were studied. Overall distributions of allele frequencies in the two groups were not significantly different. Although the I/D polymorphism in intron 18 of the NPRB gene was not associated with EH, the results of this study, which identified two novel polymorphisms in the human NPRB gene, will facilitate further genetic analysis of this gene and cardiovascular disease.

Functional deletion mutation of the 5'-flanking region of type A human natriuretic peptide receptor gene and its association with essential hypertension and left ventricular hypertrophy in the Japanese.

The natriuretic peptide (NP) family is involved in the regulation of blood pressure and fluid volume. We isolated the 5'-flanking region of the type A human NP receptor gene and identified an insertion/deletion mutation in this region. We then assessed whether there is a genetic association between this mutation and essential hypertension (EH). The deletion allele lacks 8 nucleotides and alters binding sites for the activator protein-2 (AP-2) and Zeste transcriptional factors. We genotyped 200 EH and 200 normotensive (NT) individuals and found 9 subjects with the deletion (8 in the EH group and 1 in the NT group). All 9 individuals were heterozygous. The NT subject with the mutation had left ventricular hypertrophy without hypertension. Transcriptional activity of the deletion allele was <30% that of the wild-type allele. The plasma levels of brain NP in EH patients with the deleted allele were significantly higher than the levels in the EH patients with the wild-type allele, and plasma brain NP levels were significantly higher in subjects with the deleted allele than in subjects with the wild-type allele, despite comparable blood pressures. These findings suggest that in Japanese individuals, this deletion in the human NP receptor gene reduces receptor activity and may confer increased susceptibility to developing EH or left ventricular hypertrophy.

An Insertion/Deletion Polymorphism in Intron 18 of the Type B Human Natriuretic Peptide Receptor Gene Is Not Associated with Cerebral Infarction.

The natriuretic peptide (NP) system may play a crucial role in the development of cardiovascular and renal diseases. C-type NP dilates arteries and lowers blood pressure, and it inhibits the proliferation of vascular smooth muscle cells via the type B NP receptor (NPRB). We determined and analyzed the structure of the NPRB gene and found an insertion/deletion (I/D) polymorphism in intron 18. In this experiment, we studied this I/D polymorphism in the NPRB gene in 241 subjects, including 118 patients with cerebral infarction (the CI group) and 123 control subjects (the non-CI group). Our goal was evaluate the association of this polymorphism with cerebral infarction. Our findings showed that genotype frequencies of the I/D polymorphism were in Hardy-Weinberg equilibrium. The frequencies for the II, ID, and DD alleles were 0.569, 0.374, and 0.057, respectively, in the non-CI group and 0.576, 0.356, and 0.068, respectively, in the CI group. No association was found between this polymorphism and cerebral infarction. These results suggest that this polymorphism in the NPRB gene is not linked to a pathogenic CI gene.

Nucleotide sequence of the 5′-flanking region of the type a human natriuretic peptide receptor gene and association analysis using a novel microsatellite in essential hypertension

We isolated the 5′-flanking region of the type A human natriuretic peptide receptor gene (hNPRA) using thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR). The 2.1-kb 5′ sequence upstream of the hNPRA gene lacks a TATA box but contains three potential Sp1-binding sites and an inverted CCAAT box. Moreover, we discovered a novel dinucleotide repeat marker approximately 300 bp upstream of the start codon. Using this microsatellite marker, we performed an association study in patients with essential hypertension (EH). One hundred twenty-five patients with essential hypertension and 125 age-matched subjects with normal blood pressure were studied. The number of TC/GA repeats of the three alleles ranged from 10 to 12 repeats in the 250 unrelated Japanese subjects. The allele frequency distribution in the two groups were not significantly different. Thus, the microsatellite polymorphism in the hNPRA gene is not associated with EH.

Natriuretic peptides and the acclimation of aglomerular toadfish to hypo-osmotic media.

Since the aglomerular toadfish (Opsanus tau) experiences a natriuresis following transfer to 10% seawater, we examined the role of natriuretic peptides in the acclimation of toadfish to hypo-osmotic media. Gel filtration chromatography of acid extracts of toadfish heart and kidney identified a broad peak of atrial natriuretic peptide-like immunoreactivity in both tissues, with maximal immunoreactivity in fractions coeluting with human alpha-atrial natriuretic peptide. Using a homologous bioassay to measure changes in aortic ring tension, both vasorelaxing and, surprisingly, vasoconstricting bioactivities were identified in gel fractions of heart extract. No significant vasorelaxing activity was identified in kidney extract or fractions. Instead, a potent vasoconstricting activity was observed, with maximal activity in gel fractions with an estimated MW greater than 1000 Da. Levels of atrial natriuretic peptide immunoreactivity in plasma from the caudal vein were very low in seawater toadfish and were unchanged 12 h after transfer of toadfish to 10% seawater. We conclude, that natriuretic peptides are present in the heart and kidney of toadfish. However, atrial natriuretic peptide-like peptides of cardiac origin circulating to the kidney via the caudal vein do not appear responsible for the natriuresis that ensues upon the transfer of toadfish to 10% seawater. In the absence of glomeruli, this tubular natriuresis may be regulated by natriuretic peptides present in the kidney.

Presence of Dendroaspis Natriuretic Peptide-Like Immunoreactivity in Human Plasma and Its Increase During Human Heart Failure

To determine whether Dendroaspis natriuretic peptide (DNP), a novel peptide isolated from the venom of the Dendroaspis angusticeps snake that contains a 17-amino acid disulfide ring structure similar to that in atrial, brain, and C-type natriuretic peptides, is present in normal human plasma and myocardium and whether, like the other natriuretic peptides, DNP-like immunoreactivity (DNP-LI) is activated in human congestive heart failure (CHF). Circulating DNP-LI was assessed in 19 normal human subjects and 19 patients with CHF (New York Heart Association class III or IV) with a specific and sensitive radioimmunoassay for DNP with no cross-reactivity with the other natriuretic peptides. Immunohistochemical studies that used polyclonal rabbit anti-DNP antiserum were performed on human atrial myocardial tissue obtained from four patients with end-stage CHF who were undergoing cardiac transplantation and from three donor hearts at the time of transplantation. We report that DNP-LI circulates in normal human plasma and is present in the normal atrial myocardium. In addition, DNP-LI is increased in the plasma of patients with CHF. This study demonstrates, for the first time, the presence of a DNP-like peptide in normal human plasma and in the atrial myocardium. Additionally, these studies demonstrate increased plasma DNP-LI in human CHF. These results support the possible existence of an additional new natriuretic peptide in humans, which may have a role in the neurohumoral activation that characterizes human CHF.

Organization of the Human Natriuretic Peptide Receptor A Gene

We have determined the structural organization of the human natriuretic peptide receptor A (hNPRA) gene without screening the genomic library. Based on the information for the cDNA of hNPRA, we amplified some fragments of the long polymerase chain reaction (PCR) products covering all genomic sequences of the gene and directly sequenced the products after extraction and purification. The hNPRA gene spans approximately 16 kb and contains 22 exons and 21 introns. All of the exon–intron junction sequences coincide with the GT/AG consensus sequence. The sequence encoding the transmembrane domain, one part of the hNPRA topological structure, exists in exon 7. The results of this first study, determination of the hNPRA gene structure, will facilitate further genetic analysis of the hNPRA gene and its related diseases.

Isolation and characterization of the 5'-flanking regulatory region of the human natriuretic peptide receptor C gene.

Phenotypic modulation of vascular smooth muscle cells (SMCs) plays a central role in the pathogenesis of atherosclerosis. Natriuretic peptide receptor-C (NPR-C) is highly expressed in vascular SMCs in the experimental arteriosclerotic neointimal area as well as in cultured SMCs, suggesting that increased expression of the NPR-C gene is related to the phenotypic alteration of vascular SMCs. To elucidate the molecular mechanisms and to identify the essential DNA sequences in NPR-C gene expression, a genomic clone containing over 8 kilobases of the 5'-flanking region of the human NPR-C gene has been isolated. Sequence analysis revealed that a number of putative regulatory elements including unusual tandem repeated AP-2-like sequences were observed in the 5'-flanking region. Primer extension and ribonuclease protection analyses revealed that transcription of the human NPR-C gene starts from two major regions. Promoter analysis using deletion constructs in human cells, highly producing NPR-C transcripts, showing that the region (from - 33 to + 13 relative to the transcription start point) had a potential promoter activity suggested that the region from -33 to + 13, containing a pyrimidine-rich stretch composed of four CTTTTT-repeated sequences, is sufficient for the proximal promoter activity. Moreover, three distinct DNA sequences surrounding the transcription start site (P1, from -60 to -33; P2, from + 14 to +40; P3, from +41 to +66) were revealed to be functional as a cis-acting positive enhancer, and a nuclear protein(s) from the human cells was demonstrated to specifically bind to the sequences, respectively. However, promoter analysis has shown that the P2 and P3 sequences could not activate the human NPR-C promoter in a synergistic manner. On the basis of deoxyribonuclease I footprinting analysis showing that a DNA element from +48 to +60 within the P3 sequence is preferentially protected, the P3 sequence appears to contain a potential regulatory element involved in NPR-C gene expression. The present study demonstrated the structure of the 5'-regulatory region of the human NPR-C gene and multiple cis-acting positive sequences closely located around the transcription start points with an important role in regulation of human NPR-C gene expression.

Isolation and Characterization of the 5'-Flanking Regulatory Region of the Human Natriuretic Peptide Receptor C Gene

Isolation and Characterization of the 5'-Flanking Regulatory Region of the Human Natriuretic Peptide Receptor C Gene

Renal and vascular effects of C-type and atrial natriuretic peptides in humans.

C-type natriuretic peptide (CNP) may affect renal and vascular functions differently from atrial natriuretic peptide (ANP). The objective of this study was to compare the renal and vascular actions of CNP to those of ANP in normal men. CNP or ANP (0.005, 0.01, and 0.05 microg x kg(-1) x min(-1)) were given by infusion to eight healthy volunteers. CNP caused dose-dependent increases in natriuresis (U(Na)) and in the fractional excretion of sodium (FE(Na)) with no effect on diuresis (UV), renal plasma flow, and glomerular filtration rate (GFR). Fraction of filtration (FF) increased only with the 0.05 microg x kg(-1) x min(-1) CNP dose. ANP caused larger increases in U(Na), FE(Na), and FF than CNP and also increased UV at 0.01 and 0.05 microg x kg(-1) x min(-1) and GFR at 0.05 microg x kg(-1) x min(-1). Although the ANP and CNP infusions produced similar elevation in the respective peptides plasma levels, urinary and nephrogenous guanosine 3',5'-cyclic monophosphate increased less in response to CNP than to ANP. Blood pressure, forearm blood flow, plasma renin activity, and aldosterone remained unaffected during the peptides infusion. Plasma ANP increased slightly during CNP infusion. Our data indicate a higher threshold of renal response to CNP than to ANP. In contrast to ANP, CNP probably may not act as an endocrine factor in humans.

Comparison of the Hydrolysis of the Three Types of Natriuretic Peptides by Human Kidney Neutral Endopeptidase 24.11

The degradation of 3 human natriuretic peptides by human kidney neutral endopeptidase 24.11 has been investigated. The studies revealed that hANP-28 and hCNP-22 are the preferred substrates, whereas hBNP-32 is not. The enzyme has been known to inactivate hANP-28 from cleavage at the Cys-Phe bond at the beginning of its ring structure. Analysis of the cleavage sites of each peptide indicated that the initial cleavage site of hCNP-22 is analogous to that of hANP-28. The Cys-Phe bond of hBNP-32 was insensitive to this enzymatic cleavage. We speculate that the stability of hBNP-32 may result from the insusceptibility of its Cys-Phe bond at the beginning of the ring structure.

Inhibition of goby posterior intestinal NaCl absorption by natriuretic peptides and by cardiac extracts.

Natriuretic peptides abolish active Na+ and Cl- absorption across the posterior intestine of the euryhaline goby Gillichthys mirabilis. Inhibition by eel and human natriuretic peptides is dose-dependent with the following sequence of potencies based on experimentally determined ID50 values for inhibition of short-circuit current: eel ventricular natriuretic peptide (78 nmol.l-1), eel atrial natriuretic peptide (156 nmol.l-1), human brain natriuretic peptide (326 nmol.l-1), human alpha atrial natriuretic peptide (1.05 mumol.l-1), and eel C-type natriuretic peptide (75 mumol.l-1). Natriuretic peptides also significantly increase transcellular conductance. The observed sequence of natriuretic peptide potencies is suggestive of cellular mediation by GC-A-type NP-R1 receptors in this tissue; as expected for guanylyl-cyclase-coupled NP-R1 receptors, cyclic GMP mimics the action of natriuretic peptides on the goby intestine. Crude aqueous extracts of goby atrium and ventricle inhibited short circuit current and increased tissue conductance in a dose-dependent manner. Ventricular extract was more potent than atrial extract on both a per organ and per milligram basis.

Production of polyclonal antibody specific for human natriuretic peptide receptor B

Polyclonal antibody against human natriuretic peptide receptor B (NPR-B) was produced using as immunogen a soluble chimeric protein consisting of the extracellular domain of the receptor fused with Fc portion of human IgG. The antibody was purified with protein A column, and then subjected to an adsorption of anti-Fc antibody using IgG column. The purified antibody recognized human NPR-B but not the related receptor NPR-A. The antibody inhibited C-type natriuretic peptide (CNP)-mediated intracellular cGMP accumulation in a dose-dependent manner. With regard to specific activity for the neutralization, the antibody purified with IgG column was significantly stronger than that before the adsorption step, indicating that the purification of the antibody with IgG column was extremely effective to remove the contaminating anti-Fc antibody from anti-NPR-B antibody. Western blot analysis using the purified antibody revealed that while the native NPR-B exists as an oligomer, the truncated NPR-B lacking most of its cytoplasmic domain is a monomer. This finding suggests that the cytoplasmic region may be involved in the oligomerization of the receptor. The results in this study demonstrate that soluble IgG fusion protein is very effective and useful for generating specific antibodies to the proteins expressed on cell surface.

Histamine release induced by human natriuretic peptide from rat peritoneal mast cells

We have been interested in the effects of some popular peptides on tracheal smooth muscle. Previously, we reported that atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) had dose-dependent relaxant effects on guinea pig tracheal smooth muscle. In this study, we compared the effects of ANP, BNP and CNP on histamine release from rat peritoneal mast cells. ANP and BNP were more potent than CNP, dose-dependently increasing histamine release at a concentration of 10 −7 M or higher. CNP induced histamine release at a concentration of 10 −6 M or higher. Extracellular calcium inhibited the histamine release induced by all 3 peptides. In conclusion, the effects of these 3 peptides in rat peritoneal mast cells demonstrated adverse reactions for respiratory diseases, although our previous results showed that these peptides caused relaxation of guinea pig tracheal smooth muscle. We should note that the drugs have different actions in each organ.

Preparation of mono-radioiodinated tracers for study of the in vivo metabolism of atrial natriuretic peptide in humans

In the present paper we evaluate the optimum chemical conditions for labelling atrial natriuretic peptide (ANP) and its metabolites and for preparing highly purified radiotracers which can be used for in vivo kinetic studies of ANP in humans. Synthetic alpha h1-28ANP and some hormone metabolites were iodinated with Na125I or Na131I by means of the lactoperoxidase (ANP) or the chloramine-T (ANP metabolites) technique. The biological activity of labelled ANP was tested by means of a binding study using mouse cardiac membranes. A high-performance liquid chromatography (HPLC) procedure was used to purify the labelled hormone and the principal labelled metabolites in venous plasma samples collected up to 50 min after the injection of 125I-labelled ANP from nine healthy men. The main ANP kinetic parameters were derived from the disappearance curves of the [125I]ANP, which were satisfactorily fitted by a biexponential function in all subjects. The main advantages of this tracer technique are: (1) high accuracy, allowing the identification of the metabolites produced in vivo under steady-state conditions after injection of the precursor (labelled hormone); (2) high sensitivity, allowing the detection of minimal quantities of metabolites (that cannot be identified on the basis of the integrated areas from the ultraviolet-absorbing peaks on HPLC); (3) high specificity, allowing the detection of possible in vitro artefactual generation of cleavage products of ANP using an internal labelled standard. Utilizing this tracer method, it was possible to estimate the principal parameters of ANP kinetics and also to plot the appearance curves of the labelled metabolites produced in vivo after the injection of the labelled precursor.

Production and characterization of monoclonal antibodies against human natriuretic peptide receptor-A or -B

Monoclonal antibodies (mAbs) against human natriuretic peptide receptor-A (NPR-A) or NPR-B were produced using NPR-expressing Chinese hamster ovary (CHO) cells and soluble chimeric NPRs consisting of the extracellular domain of each receptor fused to Fc region of human IgG. Three anti-NPR-A mAbs, designated as A144, A397 and A416, bound to human NPR-A but not to NPR-B, while an anti-NPR-B mAb B136 reacted with human NPR-B but not with NPR-A. Competition analysis with the anti-NPR-A mAbs revealed that two mAbs, A144 and A416, recognize an identical or the adjacent site of the receptor and that A397 is directed against another epitope. No anti-NPR-A mAb affected binding of atrial natriuretic peptide (ANP) to NPR-A, while the anti-NPR-B mAb B136 inhibited binding of C-type natriuretic peptide (CNP) to NPR-B. Inhibition of the ligand-binding by B136 is specific in that the mAb showed no effect on the binding of ANP to NPR-A. B136 also blocked CNP-mediated intracellular cGMP accumulation in NPR-B-expressing cells. These results suggest that the region recognized by B136 may be related to the ligand-binding region of NPR-B. NPR-A- and NPR-B-expressing cells were selectively detected by immunostaining using the mAbs. These findings demonstrate that the mAbs will be useful to elucidate the role of the natriuretic peptides and their receptors in normal and disease states in human.

Cyclic GMP-mediated inhibition of L-type Ca2+ channel activity by human natriuretic peptide in rabbit heart cells.

1. Effects of atrial natriuretic peptide (ANP) on the L-type Ca2+ channels were examined in rabbit isolated ventricular cells by use of whole-cell and cell-attached configurations of the patch clamp methods. ANP produced a concentration-dependent decrease (10-100 nM) in amplitude of a basal Ca2+ channel current. 2. The inactive ANP (methionine-oxidized ANP, 30 nM) failed to decrease the current. 3. 8-Bromo-cyclic GMP (300 microM), a potent activator of cyclic GMP-dependent protein kinase (PKG), produced the same effects on the basal Ca2+ channel current as those produced by ANP. The cyclic GMP-induced inhibition of the Ca2+ channel current was still evoked in the presence of 1-isobutyl-3-methyl-xanthine, an inhibitor of phosphodiesterase. ANP failed to produce inhibition of the Ca2+ channel current in the presence of 8-bromo-cyclic GMP. 4. In the single channel recording, ANP and 8-bromo-cyclic GMP also inhibited the activities of the L-type Ca2+ channels. Both agents decreased the open probability (NPo) without affecting the unit amplitude. 5. The present results suggest that ANP inhibits the cardiac L-type Ca2+ channel activity through the intracellular production of cyclic GMP and then activation of PKG.

Characterization of the hormone binding site of natriuretic peptide receptor-C

The hormone binding site of rat and human natriuretic peptide clearance receptor (NPR-C), a single transmembrane receptor, has been further refined by mutagenesis. In addition to residue 188 (rat Ala, human Ile), which completely inverts the pharmacology of the rat and human receptors [Engel et al. (1994) J. Biol. Chem. 269, 17005–17008], we report a second key residue at position 205 (rat Tyr, human Asn) which modulates affinity to a limited number of ligands. Orthologous mutation of both residues results in tighter binding for human and weaker binding for rat NPR-C. The ligand binding fold of the receptor is formed by at least the first half of the extracellular domain containing two intramolecular disulfide loops, with the two affinity-modulating residues 188 and 205 in the second loop.

Solution conformation of an atrial natriuretic peptide variant selective for the type A receptor.

Two-dimensional NMR spectroscopy has been used to characterize the solution conformation of an atrial natriuretic peptide (ANP) variant which is selective for the human natriuretic peptide receptor A (NPR-A) relative to receptor C (NPR-C). The ANP mutant, containing six substitutions, has reduced flexibility in aqueous solution relative to wild-type ANP and allows the observation of sufficient NOE connectivities for structure determination by distance geometry and restrained molecular dynamics calculations. The solution conformation is reasonably well defined, having an average backbone atom rms deviation from the average coordinates of approximately 1.1 A for residues 7-27. The structure is consistent with available functional data and shows a spatial separation between known receptor binding determinants and residues found to be outside the hormone-receptor interface.